1. Field of the Invention
This invention relates to a chemical biologically active colony stimulating factor-1 (CSF-1) that alters the chemical and/or physiological properties of this protein. More specifically, this invention relates to selective conjugation of CSF-1 to polymers to increase the circulating half-life of the protein in mammals.
2. Background Art
Colony stimulating factor-1 (CSF-1) (also known as M-CSF) is one of several proteins that are capable of stimulating colony formation by bone marrow cells plated in semisolid culture medium. CSF-1 is distinguished from other colony stimulating factors by its ability to stimulate the formation of predominantly macrophage colonies. Other CSFs stimulate the production of colonies that consist of neutrophilic granulocytes and macrophages, exclusively neutrophilic granulocytes, or neutrophilic and eosinophilic granulocytes and macrophages. A review of these CSFs has been published by Dexter, T. M., Nature (1984) 309:746, and by Vadas, M. A., J. Immunol (1983) 130:793. There is currently no routine in vivo assay that is known to be specific for CSF-1 activity.
CSF-1 has been purified from native sources (see, e.g., copending U.S. application Ser. No. 07/002400 filed Dec. 3, 1986, assigned to the same assignee, regarding immunoaffinity chromatography of native CSF-1 to enable partial amino acid determinations). CSF-1 has also been produced from recombinant DNA using two apparently related cDNA clones: (1) a "short" form that encodes a monomeric protein of 224 amino acids preceded by a 32-amino acid signal sequence (Kawasaki, E. S., et al., Science (1985) 230:292-296); and (2) a "long" form, encoding a monomeric protein of 522 amino acids, also preceded by the 32-amino acid signal sequence. The long form has been cloned and expressed by two groups, as disclosed in copending U.S. Ser. Nos. 923,067 and 039,654, filed Oct. 24, 1986 and Apr. 16, 1987, respectively, and assigned to the same assignee and incorporated herein by reference; Wong, G., et al. Science (1987) 235:1504-1509, and PCT WO87/06954 published Nov. 19, 1987. (The DNA and amino acid sequences for these two clones are shown in FIGS. 1 and 2, respectively.) Both the long and short forms of CSF-1 are described by Clark and Kamen, Science (1987) 236:1229-1237.
The long and short forms of the CSF-1-encoding DNA appear to arise from a variable splice junction at the upstream portion of exon 6 of the genomic CSF-1-encoding DNA. When CSF-1 is expressed in certain eucaryotic cells from either the long or short cDNA forms, it is secreted as a dimeric glycoprotein and appears to be variably processed at the C-terminus and/or variably glycosylated. Consequently, CSF-1 proteins of varying molecular weights are found when the reduced monomeric form is subjected to Western analysis.
The amino acid sequences of the long and short forms, as predicted from the DNA sequence of the isolated clones and by their relationship to the genomic sequence, are identical in the first 149 amino acids at the N-terminus after signal peptide cleavage, and diverge thereafter as a result of the insertion in the longer clone of an additional 894 bp fragment (encoding 298 additional amino acids) before the codon encoding amino acid 150. Therefore, both the shorter and longer forms of the gene encode regions of identical sequence at the C-terminus, as well as at the N-terminus. Biologically active protein has been recovered when truncated cDNAs encoding only the first 150 or 158 amino acids of the mature short form, or the first 190 or 221 amino acids of the mature longer form, are expressed in eucaryotic cells.
Recombinant CSF-1 was expressed in E. coli by modifying a short clone cDNA originally described by Kawasaki et al., Science (1985) 230:291 to code for proteins that contained (1) the native N-terminus and a C-terminus at amino acid 150 of the mature protein, and (2) a truncation to delete the first two amino acids at the N-terminus and a C-terminus at amino acid 150 of the mature protein. These proteins were purified and refolded to form homodimers and were found to have apparent molecular weights on size-exclusion high performance liquid chromatography (HPLC) of about 43,000 and 40,000 daltons, respectively. CSF-1 proteins modified so that the C-terminus of the expressed protein is amino acid 150 or 158 and so that up to three amino acids at the N-terminus are deleted have also been prepared.
Small proteins (less than about 70 kd) often have a relatively short half-life in blood after intravenous injection. Rapid clearance of drugs from circulation often reduces their efficacy. It is often desirable to increase the half-life of a circulating polypeptide so that smaller amounts of the polypeptide or less frequent injections might be administered, while retaining the desired therapeutic effect. Modifications of the CSF-1 protein that might alter its half-life in vivo, reduce its immunogenicity, or reduce or eliminate aggregation of the protein that might occur when it is introduced in vivo would be desirable. Such modifications include the modification of proteins with substantially straight chain polymers such as polyethylene glycol (PEG), polypropylene glycol (PPG), dextran, or polyvinyl alcohol.
For example, U.S. Pat. No. 4,261,973 describes conjugation of immunogenic allergen molecules with non-immunogenic water-soluble polymers such as PEG or polyvinyl alcohol to reduce the immunogenicity of the allergen. U.S. Pat. No. 4,301,144 describes conjugation of hemoglobin to PEG, PPG, a copolymer of ethylene glycol with propylene glycol, or ethers, esters or dehydrated products of such polymers to increase the oxygen-carrying ability of the hemoglobin molecule. U.S. Pat. No. 4,609,546 discloses that conjugation of a polypeptide or glycoprotein such as a colony stimulating factor to a polyoxyethylenepolyoxypropylene copolymer may increase the duration of its physiological activity. The only proteins that have been tested in this fashion are enzymes or native interferon, which are readily water-soluble. PCT WO 86/04145 published July 17, 1986 discloses PEG modification of antibodies to decrease binding to Fc receptors. U.S. Pat. No. 4,179,337 discloses conjugation of water-soluble polypeptides such as enzymes and insulin to PEG or PPG to reduce the immunogenicity of the polypeptides while retaining a substantial proportion of their desired physiological activities. EP No. 154,316, published Sept. 11, 1985 to Takeda Chemical Industries, Ltd., discloses and claims chemically modified lymphokines such as IL-2 containing PEG bonded directly to at least one primary amino group of the lymphokine. In addition, Katre et al., Proc. Natl. Acad. Sci. (1987) 84:1487 discloses modification of IL-2 with PEG.
Many other references disclose the concept of PEG derivatization of proteins such as alpha-1-proteinase inhibitor, asparaginase, uricase, superoxide dismutase, streptokinase, plasminogen activator, IgG, albumin, lipoprotein lipase, horseradish peroxidase, catalase, arginase and asparaginase, as well as peptides. Such derivatization through lysines was reported as improving half-life, decreasing immunogenicity, increasing solubility, and in general, increasing efficacy (which permitted less frequent dosing). In most cases, the proteins required multiple modifications per molecule to achieve improved performance in vivo, and the activity in vitro was significantly decreased by such modification.
Modification of IL-2, IFN-.beta. and immunotoxins with PEG through cysteine residues of a polypeptide is disclosed in PCT WO87/00056 published Jan. 15, 1987.
Copending U.S. patent application Ser. No. 053,244 filed May 22, 1987 discloses active ester forms of poly(alkylene glycols) that do not contain an ester linkage between the poly(alkylene glycol) and the terminal carboxylic acid and can be reacted with proteins in a controlled and reproducible manner.
In addition to these patents and patent publications, several articles discuss the concept of using activated PEG or PPG as a modifying agent for proteins such as enzymes, IgG and albumin. For example, Inada et al., Biochem. and Biophys. Res. Comm., 122, 845-850 (1984) disclose modifying water-soluble lipoprotein lipase to make it soluble in organic solvents such as benzene by using cyanuric chloride to conjugate with PEG. Takahashi et al., Biochem. and Biophys. Res. Comm., 121:261-265 (1984) disclose modifying horseradish peroxidase using cyanuric chloride triazine with PEG to make the water-soluble enzyme active and soluble in benzene.
Patents and patent publications that disclose use of polyvinyl alcohol (PVA) in protein conjugation reactions include U.S. Pat. Nos. 4,296,097 and 4,430,260, relating to conjugation of benzylpenicillin and PVA, U.S. Pat. No. 4,496,689 (EP No. 147,761), relating to conjugation of alpha-1-proteinase inhibitor with a polymer such as heparin, PVA or PEG, EP No. 142,125 published May 22, 1985, disclosing non-covalent bonding of hemoglobin to PVA as a carrier, DE No. 2312615 (Exploaterings AB TBF), relating to crosslinked, water-insoluble PVA coupled to a protein, and DE No. 3,340,592 published May 23, 1985, relating to conjugates of PVA with human hemoglobin A.
Articles relating to conjugates of proteins and PVA include Sabet et al., Indian J. Chem., Sec. A (1984) 23A(5) (disclosing PVA and protein interaction), Wei et al., Immunol. (1984) 51(4):687-696 (disclosing trimellityl conjugated with PVA), Lee et al., J. Immunol. (1981) 126:414-418 and Hubbard et al., J. Immunol. (1981) 126:407-413 (both disclosing DNP conjugated to PVA), Lee et al., Int. Arch. Allergy Appl. Immunol. (1980) 63:1-13 (disclosing antibenzylpenicilloyl IgE conjugated to PVA), Sehon, Prog. Allergy (1982) 32:161-202 (disclosing an allergen and hapten conjugated via PVA), Holford-Strevens et al., Int. Arch. Allergy App. Immunol. (1982) 67:109-116 (disclosing conjugation of PVA and an antigen/hapten), and Sehon and Lee, Int. Arch. Allergy App. Immunol. (1981) 66 (Supp. 1), pp. 39-42 (disclosing a hapten/allergen conjugated to PVA).
Copending U.S. application Ser. No. 099,872 filed Sept. 22, 1987 discloses various potential uses of CSF-1, including use as an anti-infection, anti-tumor, or wound-healing agent.
None of these references, however, discloses details of how to modify CSF-1 with a polymer such as PEG or polyvinyl alcohol so as to retain its biological activity while also increasing its circulating half-life or efficacy. Furthermore, it is not generally possible to predict the extent of protein modification that is desirable, because some proteins are much more susceptible to inactivation through conjugation than others.